摘要
目的：间充质干细胞是普遍存在于人体各组织中的一种成体干细胞,可从骨髓、外周血、脐血、脂肪组织、骨外膜、骨小梁、肌肉、胎肺、胎肾等组织中分离出来。实验观察了人胎儿骨髓间充质干细胞体外分离和培养的方法,观察其生物学特性,以及向各胚层分化的可行性。方法：实验于2006-07/2007-08在苏州大学附属第二医院血液科实验室完成。①实验材料：4个月胎龄的流产胎儿由苏州大学附属第二医院妇产科提供,产妇及家属均签署知情同意书,实验经医学伦理委员会批准。②实验方法：采用Ficoll法分离胎儿骨髓腔冲洗液,离心后取单核细胞层培养、扩增、传代,观察细胞生物学特性,采用MTT法观察细胞生长曲线及分裂指数曲线。取第3代细胞,在含新生牛血清、地塞米松、β-甘油磷酸钠、抗坏血酸的DMEM-LG培养基中诱导其成骨分化;在含新生牛血清、地塞米松、胰岛素的DMEM-LG培养基中诱导其成脂分化;在含bFGF、N2、DMEM-LG培养液中诱导其成神经分化。③实验评估：于3周后行vonKossa染色、油红O染色和免疫组织化学检测Nestin、NES、NF的表达以鉴定诱导后的细胞。结果：①接种48h后,贴壁细胞呈梭形、多角形、成纤维细胞样形态。1周后开始传代,传代培养的潜伏期为24～36h,对数增殖期约为二三天,第6天进入平台期。传代至8代后生长速度变慢。②FCM检测显示,细胞高表达CD29、CD44及CD49e分子,低表达CD106、CD54、CD11b,而不表达CD34。③成骨、成脂培养基诱导3周后,Von-Kossa染色呈黑色,油红染色成橘红色。诱导后的胎儿骨髓间充质干细胞于第4天进行免疫组织化学染色显示Nestin、NF、NES表达阳性。结论：采用Ficoll密度梯度离心、贴壁筛选法及单克隆培养法,分离、培养、扩增出具有高度同源性的胎儿骨髓间充质干细胞,并在体外成功的诱导其向成骨细胞、脂肪细胞和神经元细胞分化。
AIM： Mesenchymal stem cells generally reside in all tissues of human body, which can be isolated from bone marrow, peripheral blood, cord blood, adipose tissue, periosteum, bone trabecula, muscle, fetus lung, fetus kidney and so on. This study explored the condition of the isolation and culture of fetal bone marrow mesenchymal stem cells in vitro. We observed both the biological characteristics of fetal bone marrow mesenchymal stem cells and the potential of differentiating into adipocytes, ostroblasts and neurons. METHODS： Experiments were conducted at Laboratory of Hematology of Second Affiliated Hospital of Soochow University from July 2006 to August 2007. （1）Four-month-old fetus samples were provided by Department of Gynaecology and Obstetncs of Second Affiliated Hospital of Soochow University. The puerperant and her relatives all signed informed consent for extraction. The experiment was authorized by Medical Ethics Committee. （2）Fetal bone marrow mesenchymal stem cells were isolated from bone marrow blood by the Ficoll method. Monocytes were cultured, amplified and passaged after centrifugation to observe the biological feature of cells. Growth curve and divisional index curve of fetal bone marrow mesenchymal stem cells were detected by MTT. Third passage cells were differentiated into ostroblasts in the DMEM-LG medium containing new-born calf serum, dexamethasone, β -glycerophosphate, antiscorbic acid, into adipocytes in the DMEM-LG medium containing new-born calf serum, dexamethasone, insuline, into neurons in the DMEM-LG medium containing bFGF and N2. （3）Three weeks later, fetal bone marrow mesenchymal stem cells was stained by oil red and von Kossa and Nestin, NES and NF was detected by immunohistochemical method in order to identify the cultured cells. RESULTS： （1）After seeded 48 hours, the adherent cells showed spindle shape, polygonal shape and fibroblast-cell-like shape. Fetal bone marrow mesenchymal stem cells were passaged after one week. The fetal bone marrow mesenchymal stem cells were still in latent phase after being passaged for 24-36 hours. 2-3 days later, cells were in log phase; 6 days later, cells came into platform phase, Until 8 passages, the growth rate of fetal bone marrow mesenchymal stem cells decreased. （2）Flow cytometry showed that fetal bone marrow mesenchymal stem cells expressed highly CD29, CD44 and CD49e, and expressed low level of CD106, CD54, CD 11b, but fetal bone marrow mesenchymal stem cells did not express CD34. （3）Fetal bone marrow mesenchymal stem cells which cultured in osteogenic inducer could change into black when stained by von Kossa, and fetal bone marrow mesenchymal stem cells which cultured in adipogenetic inducer could become jacinth when stained by oil red. Neuron-like cells expressed Nestin, NF and NES markers by immunohistochemical method at day 4. CONCLUSION： Fetal bone marrow mesenchymal stem cells with high homology are isolated, cultured, proliferated by Ficoll density gradient centrifugation, attachment method and monoclone cultivation. Fetal bone marrow mesenchymal stem cells can be induced into adipocytes, ostroblasts and neuron-like cell successfully in vitro.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2008年第12期2253-2257,共5页
Journal of Clinical Rehabilitative Tissue Engineering Research
基金
国防预研(616010305)
苏州市社会发展基金(SS0534)资助项目~~
